<p> Supplementary Tables</p><p>Supplementary Table S1. Clinicopathological Characteristics. Characteristic Number of patients (%) Age, median(range) 61(38-84)years Gender Male 143(77.3) Female 42(22.7) Smoking status Never 88(47.6) Former/current 97(52.4) Surgical approach Sweet approach 130(70.3) Ivor Lewis approach 34(18.4) Ivor Lewis approach+cervical incision 16(8.6) Sweet approach+cervical incision 2(1.1) Minimally invasive esophagectomy 3(1.6) Tumor stage Ⅰ A 3(1.6) Ⅰ B 10(5.4) Ⅱ A 50(27.0) Ⅱ B 45(24.3) Ⅲ A 44(23.8) Ⅲ B 23(12.4) Ⅲ C 10(5.4) Tumor differentiation Well/moderate 105(56.8) Poor 80(43.2) Charlson comorbidity index (CCI) <3 138 (74.6) ≥3 47 (25.4) Note: Tumor stage was determined according to 7th edition of UICC-AJCC TNM staging system</p><p>Supplementary Table S2. Differently expressed miRNAs between ECs and NMs. miRNA Sequence ECs vs.NMs</p><p>1 Log2FC Style P-Value FDR hsa-miR-126-3p tcgtaccgtgagtaataatgcg -1.647483494 down 0 0 hsa-miR-451a aaaccgttaccattactgagtt -1.65895591 down 0 0 hsa-miR-99b-5p cacccgtagaaccgaccttgcg -1.66539343 down 0 0 hsa-miR-26a-5p ttcaagtaatccaggataggct -1.683017076 down 0 0 hsa-miR-26b-5p ttcaagtaattcaggataggt -1.709578252 down 0 0 hsa-miR-126-5p cattattacttttggtacgcg -1.816966633 down 0 0 hsa-miR-100-5p aacccgtagatccgaacttgtg -1.824441371 down 0 0 hsa-miR-10a-5p taccctgtagatccgaatttgtg -1.867781043 down 0 0 hsa-miR-143-5p ggtgcagtgctgcatctctggt -1.892258222 down 0 0 hsa-miR-143-3p tgagatgaagcactgtagctc -2.197916019 down 0 0 hsa-miR-195-5p tagcagcacagaaatattggc -2.217941529 down 0 0 hsa-miR-1 tggaatgtaaagaagtatgtat -2.351785345 down 0 0 hsa-miR-133a tttggtccccttcaaccagctg -2.74123279 down 0 0 hsa-miR-145-5p gtccagttttcccaggaatccct -2.968242676 down 0 0 hsa-miR-145-3p ggattcctggaaatactgttct -3.024082135 down 0 0 hsa-miR-139-5p tctacagtgcacgtgtctccagt -2.226581077 down 1.07E-261 8.90E-260 hsa-miR-4454 ggatccgagtcacggcacca -1.740270003 down 4.18E-217 3.08E-215 hsa-miR-125a-5p tccctgagaccctttaacctgtga -1.827815583 down 2.49E-205 1.74E-203 hsa-miR-375 tttgttcgttcggctcgcgtga -1.915837949 down 1.06E-169 6.36E-168 hsa-miR-548a-5p aaaagtaattgcgagttttacc -2.523150543 down 4.77E-148 2.66E-146 hsa-miR-4328 ccagttttcccaggatt -2.697970737 down 1.07E-141 5.83E-140 hsa-miR-28-5p aaggagctcacagtctattgag -1.601477789 down 1.62E-129 8.41E-128 hsa-miR-218-5p ttgtgcttgatctaaccatgt -1.936428345 down 2.01E-114 9.65E-113 hsa-miR-136-3p catcatcgtctcaaatgagtct -1.759250561 down 1.70E-66 6.11E-65 hsa-miR-3195 cgcgccgggcccgggtt -1.673316592 down 1.81E-60 6.01E-59 hsa-miR-4741 cgggctgtccggaggggtcggct -1.858183604 down 4.58E-52 1.35E-50 hsa-miR-335-5p tcaagagcaataacgaaaaatgt -1.59031393 down 8.31E-49 2.39E-47 hsa-miR-3681-3p acacagtgcttcatccactact -2.078739976 down 4.14E-45 1.13E-43 hsa-miR-133b tttggtccccttcaaccagcta -2.172907377 down 2.76E-42 7.34E-41 hsa-miR-4532 ccccggggagcccggcg -1.760169355 down 2.14E-41 5.60E-40 hsa-miR-378i actggactaggagtcagaagg -2.058668597 down 4.76E-36 1.15E-34 hsa-miR-10b-3p acagattcgattctaggggaat -2.24764374 down 2.27E-33 5.14E-32 hsa-miR-337-5p gaacggcttcatacaggagtt -1.974437863 down 8.17E-31 1.71E-29 hsa-miR-4455 agggtgtgtgtgttttt -2.024008714 down 2.50E-21 3.86E-20 hsa-miR-10a-3p caaattcgtatctaggggaata -1.966466764 down 8.89E-21 1.34E-19 hsa-miR-4770 tgagatgacactgtagct -2.038610548 down 1.84E-20 2.71E-19 hsa-miR-4509 actaaaggatatagaaggtttt -2.397678635 down 6.57E-17 8.55E-16 hsa-miR-4745-3p tggcccggcgacgtctcacggtc -1.736815844 down 4.60E-16 5.76E-15 hsa-miR-4638-3p cctggacaccgctcagccggccg -1.692441591 down 2.00E-12 2.08E-11 hsa-miR-378h actggacttggtgtcagatgg -1.603674809 down 2.13E-07 1.47E-06 hsa-miR-4691-3p ccagccacggactgagagtgcat -1.743000579 down 5.33E-06 3.07E-05 hsa-miR-376b-5p cgtggatattccttctatgttt -1.648405924 down 0.0011277 0.0042316</p><p>2 hsa-miR-302a-3p taagtgcttccatgttttggtga -1.951900185 down 0.005660 0.017287 8 hsa-miR-302b-3p taagtgcttccatgttttagtag -1.702520189 down 0.016806 0.0421777 1 hsa-miR-7-5p tggaagactagtgattttgttgt 2.022229924 up 0 0 hsa-miR-21-3p caacaccagtcgatgggctgt 1.843075582 up 0 0 hsa-miR-5588-5p actggcattagtgggactttt 1.829174581 up 0 0 hsa-miR-141-3p taacactgtctggtaaagatgg 1.806832619 up 0 0 hsa-miR-96-5p tttggcactagcacatttttgct 1.787574712 up 0 0 hsa-miR-3529-3p aacaacaaaatcactagtcttcca 2.099750195 up 4.53E-262 3.79E-260 hsa-miR-224-5p caagtcactagtggttccgtt 2.061727489 up 1.91E-242 1.52E-240 hsa-miR-18a-5p taaggtgcatctagtgcagatag 1.9798533 up 5.75E-190 3.86E-188 hsa-miR-135b-5p tatggcttttcattcctatgtga 2.328348343 up 8.72E-185 5.74E-183 hsa-miR-34c-5p aggcagtgtagttagctgattgc 2.381382549 up 9.93E-170 5.98E-168 hsa-miR-1260b atcccaccactgccaccat 1.645866709 up 3.66E-122 1.86E-120 hsa-miR-182-5p tttggcaatggtagaactcacact 1.662900442 up 5.55E-96 2.44E-94 hsa-miR-200a-5p catcttaccggacagtgctgga 1.701196601 up 6.22E-76 2.36E-74 hsa-miR-196a-5p taggtagtttcatgttgttggg 1.889754261 up 1.02E-72 3.83E-71 hsa-miR-944 aaattattgtacatcggatgag 1.747372199 up 6.25E-59 2.04E-57 hsa-miR-4533 tggaaggaggttgccggacgct 2.524792369 up 1.85E-52 5.49E-51 hsa-miR-1305 ttttcaactctaatgggagaga 2.040124142 up 4.42E-30 9.14E-29 hsa-miR-3136-5p ctgactgaataggtagggtcatt 1.786858781 up 6.27E-28 1.21E-26 hsa-miR-18b-5p taaggtgcatctagtgcagttag 1.955033645 up 9.23E-26 1.66E-24 hsa-miR-3978 gtggaaagcatgcatccagggtg 1.611424083 up 6.54E-25 1.14E-23 t hsa-miR-1269a ctggactgagccgtgctactgg 1.821194054 up 1.95E-22 3.08E-21 hsa-miR-562 aaagtagctgtaccatttgc 1.824669665 up 5.84E-22 9.12E-21 hsa-miR-5687 ttagaacgttttagggtcaaat 2.12039056 up 1.31E-19 1.86E-18 hsa-miR-18a-3p actgccctaagtgctccttctgg 1.83199315 up 5.09E-13 5.44E-12 hsa-miR-135b-3p atgtagggctaaaagccatggg 1.757722262 up 3.44E-12 3.51E-11 hsa-miR-4643 gacacatgaccataaatgctaa 1.730131518 up 8.76E-12 8.69E-11 hsa-miR-3975 tgaggctaatgcactacttcac 2.098725147 up 9.20E-12 9.11E-11 hsa-miR-6512-5p taccattagaagagctggaaga 1.938090549 up 2.13E-10 1.92E-09 hsa-miR-539-5p ggagaaattatccttggtgtgt 1.643218724 up 6.95E-09 5.55E-08 hsa-miR-518d-3p caaagcgcttccctttggagc 1.766620338 up 8.25E-08 5.97E-07 hsa-miR-888-5p tactcaaaaagctgtcagtca 1.631917707 up 8.85E-08 6.37E-07 hsa-miR-5197-5p caatggcacaaactcattcttga 2.044739998 up 1.23E-06 7.67E-06 hsa-miR-4759 taggactagatgttggaatta 1.811917848 up 6.08E-05 3.00E-04 hsa-miR-5692b aataatatcacagtaggtgt 1.773095835 up 0.003528 0.0115246 7</p><p>3 Supplementary Table S3. Univariate and Multivariate analysis of factors associated with disease- specific survival.</p><p>Variables Univariate Analysis Multivariate Analysis HR 95% CI P HR 95% CI P Gender (female vs. 0.791 0.494-1.267 0.329</p><p> male) Age(≥60 vs. <60) 1.044 0.709-1.538 0.826 Smoking status 0.734 0.498-1.082 0.118</p><p>(non-smokers vs. </p><p> smokers) Tumor 1.245 0.846-1.833 0.266</p><p> differentiation(Poor </p><p> vs. Well/Moderate) Surgical approach 0.127 Ivor Lewis 1.179 0.720-1.932 0.512</p><p> approach vs. Sweet </p><p> approach Other approach * 0.510 0.246-1.057 0.070</p><p> vs. Sweet approach CCI(≥3 vs. <3) 1.522 1.003-2.310 0.049 1.749 1.146-2.668 0.009 Tumor stage (Ⅲ-IV 2.567 1.736-3.795 <0.001 2.573 1.732-3.824 <0.001</p><p> vs. Ⅰ-Ⅱ) MiRNA-126 1.607 1.090-2.370 0.017 1.554 1.050-2.301 0.028</p><p>(negative vs. </p><p> positive) Abbreviations and Note: 95%CI, 95% confidence interval; CCI, Charlson comorbidity index; Other approach include Ivor Lewis approach+cervical incision, Sweet approach+cervical incision and Minimally invasive esophagectomy. </p><p>Supplementary Table S4. A short list of functionally relevant genes significantly downredulated in 126- EC cells vs. NC-EC cells.</p><p>G e n e S y m b o l Gene Description miR-126/control TargetScan Pictar miRanda miRDB</p><p> style p-</p><p>4 value(<0.05)</p><p>ADAM9 ADAM metallopeptidase domain 9 down 6.68E-58 + + + +</p><p>PIK3R2 phosphoinositide-3-kinase, regulatory subunit 2 (beta) down 1.67E-37 - - + -</p><p>SYDE1 synapse defective 1, Rho GTPase, homolog 1 (C. elegans) down 7.12E-17 - - + -</p><p>GPR64 G protein-coupled receptor 64 down 8.87E-07 - - - +</p><p>CHST11 carbohydrate (chondroitin 4) sulfotransferase 11 down 1.24E-05 - - + -</p><p>TMEM128 transmembrane protein 128 down 1.63252E-05 - - - +</p><p>IGSF9B immunoglobulin superfamily, member 9B down 5.09E-05 - - + -</p><p>OPLAH 5-oxoprolinase (ATP-hydrolysing) down 5.83E-05 - - + -</p><p>PAG1 phosphoprotein associated with glycosphingolipid microdomains down 7.27E-05 - - + -</p><p>1</p><p>LARP6 La ribonucleoprotein domain family, member 6 down 0.000283627 - - + -</p><p>KSR1 kinase suppressor of ras 1 down 0.001202221 - - + +</p><p>PIK3CD phosphoinositide-3-kinase, catalytic, delta polypeptide down 0.001629352 - - + -</p><p>CALHM3 calcium homeostasis modulator 3 down 0.001769405 - - - +</p><p>IGF2BP2 insulin-like growth factor 2 mRNA binding protein 2 down 0.00193895 - - + -</p><p>ZXDA zinc finger, X-linked, duplicated A down 0.002413057 - - - +</p><p>MYH11 myosin, heavy chain 11, smooth muscle down 0.005472495 - - + -</p><p>ZBTB7C zinc finger and BTB domain containing 7C down 0.005873784 - - - +</p><p>ZC3H12B zinc finger CCCH-type containing 12B down 0.006536688 - - + +</p><p>LRP1B low density lipoprotein receptor-related protein 1B down 0.011267139 - - - +</p><p>SLC12A3 solute carrier family 12 (sodium/chloride transporters), member 3 down 0.013367434 - - + -</p><p>TMEM40 transmembrane protein 40 down 0.014538934 - - + -</p><p>HIST1H2BN histone cluster 1, H2bn down 0.018131111 - - - +</p><p>APC2 adenomatosis polyposis coli 2 down 0.020848075 - - + -</p><p>ZNF333 zinc finger protein 333 down 0.020848075 - - - +</p><p>RAPGEF3 Rap guanine nucleotide exchange factor (GEF) 3 down 0.02449727 - - - +</p><p>TCL6 T-cell leukemia/lymphoma 6 (non-protein coding) down 0.024841435 - - + -</p><p>UCN2 urocortin 2 down 0.027078987 - - + -</p><p>PIGR polymeric immunoglobulin receptor down 0.027964082 - - + -</p><p>SOBP sine oculis binding protein homolog (Drosophila) down 0.043127863 - - + -</p><p>GJB2 gap junction protein, beta 2, 26kDa down 0.043433224 - - - +</p><p>ENO4 enolase family member 4 down 0.047004076 - - - +</p><p>5 +: genes presented as potential targets of miR-126 in TargetScan, PicTar, miRanda or miRDB. Down: genes downregulated in miR-126 overexpressed EC-109 cells vs. control EC-109 cells.</p><p>Supplementay Table S5. Primer sequences (5’-3’).</p><p>6 PCR primers for Methylation-specific PCR (MSP) methylated forward GGGTTTGAAAGTTAGTAAAGGGTT Egfl7 reverse GCGACCCGCCAACACC Unmethylated forward TGGGGGGTTTGAAAGTTAGTAA Egfl7 reverse ACCTCTAAACCACAACCCAC Primers for ADAM9-UTR and DNMT1-UTR clones ADAM9 forward GCTCTAGAGTATTCACGCAGTTACTCGCT</p><p> reverse TATAGGCCGGCCCTTCCTTCTTTCTCCAGCCT</p><p>DNMT1 forward GCTCTAGA CTCCCGTCACCCCTGT</p><p> reverse TATAGGCCGGCCAAGTCTTAATTTCCACTCATACAG Primers for real time PCR</p><p>ADAM9 forward GCTAGTTGGACTGGAGATTTGG</p><p> reverse GTGGCTCCTTGAACACACTG</p><p>DNMT1 forward TCTACCAGGGAGAAGGACAGG</p><p> reverse CTCACAGACGCCACATCGC</p><p>PIK3R2 forward AATGCAGCAAGGAATACCTGG</p><p> reverse GCTCTCATGGATCTCGGCAA</p><p>VEGFA forward CAGAAGGAGGAGGGCAGAAT</p><p> reverse CTCGATTGGATGGCAGTAGC</p><p>IRS1 forward CTGCACAACCGTGCTAAGG</p><p> reverse CGTCACCGTAGCTCAAGTCC</p><p>Crk forward GCGGAGTAGCTGGTACTGG</p><p> reverse GCGCGAGTTCTCTGAGACG</p><p>GAPDH forward TGCACCACCAACTGCTTAGC</p><p> reverse GGCATGGACTGTGGTCATGAG siRNA sequence si-DNMT1 sense CGAGUUGCUAGACCGCUUCTT </p><p> antisense GAAGCGGUCUAGCAACUCGTT si-ADAM9-1 sense CCAAUGGAAACCUGAUCAA dTdT (S1) antisense dTdT GGUUACCUUUGGACUAGUU si-ADAM9-2 sense CCAGAAUAACAAAGCCUAU dTdT (S2) antisense dTdT GGUCUUAUUGUUUCGGAUA si-ADAM9-3 sense GCGAAGGAAGUACCUGUAA dTdT (S3) antisense dTdT CGCUUCCUUCAUGGACAUU</p><p>7 Supplementary Table S6. The primary antibodies for western blot and IHC.</p><p>Antibody Concentration Concentration Specificity Company Catalog #</p><p> for WB for IHC</p><p>DNMT1 1:1000 / Rabbit monoclonal CST 5032S</p><p>PIK3R2 1:1000 / Rabbit polyclonal Abcam ab71052</p><p>ADAM9 1:1000 / Rabbit polyclonal CST 2099S</p><p>/ 1:100 Rabbit polyclonal Abcam ab186833</p><p>PEGFR 1:1000 / Rabbit monoclonal Epitomics 1727-1</p><p>(Abcam) (ab40815)</p><p>EGFR 1:1000 / Rabbit monoclonal Epitomics 2116-1</p><p>(Abcam) (ab76153) PAKT 1:1000 / Rabbit monoclonal Epiomics 2118-1</p><p>(Abcam) (ab81283) AKT 1:1000 / Rabbit monoclonal Epitomics 1085-1</p><p>(Abcam) (ab32038)</p><p>HRP-GAPDH 1:10000 / Mouse monoclonal KangChen KC-5G5</p><p>Abbreviations: WB, western blot; IHC, immunohistochemistry; CST, Cell Signaling Technology </p><p>8</p>